Biomedical subjects

J Melle

Publications and source records attributed to J Melle.

At least 19 recordsLinked to original sources

PubMed2001

Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation.

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Blood Platelets↗

PubMed2001

Flow cytometry detection of platelet procoagulation activity and microparticles in patients with unstable angina treated by percutaneous coronary angioplasty and stent implantation.

Platelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and microparticles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p < 0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.

Aged↗

PubMed1999

Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction.

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)-induced BFU-E and CFU-E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo-R in MDS marrow, we focused on Epo-induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro-apoptotic signals rather than the defect of anti-apoptotic pathways resulting from Epo-stimulated Jak2-STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.

Adult↗

PubMed1998

Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia.

Thrombocytopenia is a frequent feature of myelodysplastic syndromes (MDS) that could be improved by the use of recombinant human megakaryocyte growth and development factor (rHuMGDF). Using short-term liquid cultures and progenitor assays, we have found that rHuMGDF stimulated DNA synthesis and potentiated leukemic cluster growth of bone marrow mononuclear cells in 10/38 MDS cases (26%). Cytogenetically malignant colonies were detectable in rHuMGDF-stimulated cultures (n=3) by fluorescence in situ hybridization. rHuMGDF was able to stimulate CFU-MK formation in 45% of the samples tested. Finally, rHuMGDF-induced blast cell proliferation correlated with elevated expression of c-MPL, previously identified as a bad prognosis factor in MDS.

Blast Crisis↗

PubMed1998

Elevated thrombopoietin serum concentrations in myelodysplasias.

Thrombopoietin (TPO) serum levels were quantified in patients with myelodysplastic syndromes using an ELISA test for TPO. We found that TPO levels were significantly elevated in the whole group of patients as compared with normal healthy donors (521.9 75.3 pg/ml vs 160.1 19.6 pg/ml; P = 0.011). TPO serum levels were inversely correlated with the megakaryocyte mass in both the RA/RARS and RAEB/RAEBt subgroups ( P = 0.012 and P = 0.031, respectively). Two subsets of patients with a possible dysregulation of TPO production were identified.

Journal Article↗

PubMed1998

Thrombopoietin activates human platelets and induces tyrosine phosphorylation of p80/85 cortactin.

The mechanism of human platelet activation by thrombopoietin (TPO) was investigated in vitro. We found that rHuTPO stimulated thromboxane A2 formation and serotonin secretion, despite the absence of shape change and aggregation. Blockade of the arachidonic acid pathway did not inhibit rHuTPO-induced platelet secretion. rHuTPO stimulated the tyrosine phosphorylation of 64, 80/85, 95, 130 and 140 kDa proteins, but phosphoproteins of 100-105 and 125 kDa obtained when platelets aggregated in the presence of thrombin were absent. rHuTPO stimulated and potentiated the thrombin-induced tyrosine phosphorylation of a 80 kDa protein identified as the cortical actin-associated protein, p80/85 cortactin. When platelets were aggregated in the presence of rHuTPO and fibrinogen, cortactin phosphorylation was enhanced as compared to rHuTPO alone. Treatment with RGDS or cytochalasin D respectively reduced or abolished cortactin tyrosine phosphorylation. This confirms the existence of fibrinogen binding-dependent and independent pools of phosphorylated cortactin, both requiring intact actin polymerization. Cytoskeleton-binding proteins may be implicated in in vitro platelet activation by rHuTPO.

Blood Platelets↗

PubMed1997

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias.

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15-47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.

Adult↗

PubMed1997

Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes.

Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% +/- 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% +/- 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.

Adult↗

PubMed1996

Thrombocytemia and abnormal megakaryopoiesis associated with abnormality of chromosome 1p34 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by a cytopenia related to abnormal proliferation and differentiation of marrow precursor cells. Some subtypes of MDS are associated with thrombocytemia or abnormal megakaryopoiesis and certain specific karyotypic abnormalities. We report on four cases of MDS with normal or elevated blood platelet count and a recurring abnormality in chromosome 1p34. The gene involved appears to be different from c-mpl and TPO.

Aged↗

PubMed1995

Detection of the chromosome 16 CBF beta-MYH11 fusion transcript in myelomonocytic leukemias.

Karyotypic detection of chromosomal 16 abnormalities classically associated with AML M4Eo can be difficult. Characterization of the two genes involved in the inv(16)(p13q22), CBF beta and MYH11, has allowed the detection of fusion transcripts by reverse-transcriptase polymerase chain reaction (RT-PCR). We have analyzed CBF beta-MYH11 fusion transcripts by RT-PCR in myelomonocytic leukemias, with or without eosinophilia, to determine whether their presence correlates with morphology. Fifty-three cases (11 AML M4Eo; 1 AML M4 with atypical abnormal eosinophils (AML M4 "Eo"); 29 AML M4; 8 AML M5; 3 CMML; and 1 AML M2 with eosinophilia) were analyzed. All 11 typical AML M4Eo were CBF beta-MYH11 positive. The single case of AML M4 with distinctive eosinophil abnormalities was negative by karyotype, RT-PCR and fluorescent in situ hybridization (FISH). Three of 29 (10%) AML M4 without abnormal eosinophils were CBF beta-MYH11 positive, 1 of which did not show any apparent chromosome 16 abnormalities by classical metaphase analysis (2 not tested). Both cases tested also showed MYH11 genomic rearrangement. None of the other leukemias were RT-PCR positive. Follow-up of three patient showed residual positivity in apparent complete remission. These data show that CBF beta-MYH11 fusion transcripts occur not only in the vast majority of typical AML M4Eo, but also in approximately 10% of AML M4 without eosinophilic abnormalities, a much higher incidence than the sporadic reports of chromosome 16 abnormalities in AML M4 would suggest. Taken together with the detection of CBF beta-MYH11 transcripts in the absence of apparent chromosome 16 abnormalities by classical banding techniques, these data show that additional screening by either RT-PCR or FISH should be performed in all AML M4, regardless of morphologic features, to allow accurate evaluation of the prognostic importance of this fusion transcript.

Adolescent↗

PubMed1995

Thrombopoietin is not responsible for the thrombocytosis observed in patients with acute myeloid leukemias and the 3q21q26 syndrome.

Patients with acute myeloblastic leukaemia (AML) and chromosomic abnormalities of the 3q21;q26 region have striking dysmegakaryopoiesis and normal or increased platelet counts. Leukaemic cells ectopically express the Evi-1 gene which maps to human chromosome 3q26:q27. Thrombopoietin (TPO) has been cloned recently and shown to be the major hormone stimulating both megakaryocytopoiesis and thrombopoiesis. The TPO gene maps to human chromosome 3q26. For this report we studied four patients with typical 3q21:q26 syndrome. Karyotype analysis showed inv(3)(q21;q26) in three cases and t(3:3)(q21;q26) in one case. Although high levels of Evi-1 transcripts could be detected in mRNA isolated from the bone marrow cells of these patients by Northern blot analysis, no TPO transcripts were detectable by RT-PCR technique on the same mRNA samples. These results demonstrate that TPO gene transcription is not activated in patients with 3q26 chromosomic abnormality, and that abnormal TPO production is not responsible for the observed thrombocytosis.

Acute Disease↗

PubMed1995

c-mpl expression in hematologic disorders.

The c-mpl gene encodes a member of the hematopoietic cytokine receptor superfamily. This gene was discovered through the study of a murine retrovirus which induces an acute myeloproliferative syndrome in mice. MPLV (for myeloproliferative leukemia virus) has transduced a truncated and constitutively activated form of the c-mpl receptor chain. The c-mpl ligand is unknown, but recent data indicate that it could specifically regulate thrombocytopoiesis. This review focuses on the expression of the c-mpl gene in a large series of human hematopoietic pathologies by Northern blot analysis. Barely detectable transcript levels were detected in normal bone marrow (BM) and in BM samples from chronic myeloproliferative disorders, plasmocytoma, Burkitt lymphoma or acute lymphoid leukemia. In contrast, high levels of c-mpl expression were detected in 45% of acute myeloid leukemia (AML). No correlation was found between c-mpl expression and the French-American-British classification subtype of AML. However c-mpl expression correlated with CD34 expression, and unfavorable cytogenetic abnormalities, defining a subgroup of AML with a low rate of complete remission. In myelodysplasia, c-mpl expression was elevated in 44% of chronic myelomonocytic leukemia (CMML), 42% of refractory anemia with excess myeloblasts (RAEB), and RAEB in transformation to acute leukemia (RAEBt), but not in refractory anemia (RA) and RA with ringed sideroblasts (RARS). In CMML, there was no correlation between c-mpl expression and any prognostic factor tested, nor with the course of the disease. The biologic significance of c-mpl expression in RAEB and RAEBt is probably different.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Prognostic value of c-mpl expression in myelodysplastic syndromes.

The c-mpl proto-oncogene which encodes a member of the hematopoietic cytokine receptor superfamily has been recently shown to be the receptor for thrombopoietin (TPO), which stimulates megakaryocyte progenitor expansion and differentiation. We studied c-mpl expression by Northern blot analysis, in a large series of 58 MDS. No expression was found in 14 patients with refractory anemia (RA) or with refractory anemia with ring sideroblasts (RARS). In contrast 11/26 (42%) patients with refractory anemia with excess of blasts (RAEB), or with RAEB in transformation (RAEBt), and 8/18 (44%) patients with chronic myelomonocytic leukemia (CMML) expressed c-mpl. In CMML patients, no correlation was found between c-mpl expression and any prognostic factor tested, nor with the course of the disease. In contrast, in RAEB and RAEBt, expression of c-mpl was correlated with high Bournemouth scoring (P < 0.005) and poor survival (P = 0.02) due to leukemic transformation. Forty-five per cent (5/11) of the c-mpl positive patients evolved towards AML with a mean follow-up of 10.5 months, while 13% (2/15) of the c-mpl negative patients developed a secondary leukemia, with a mean follow-up of 21.1 months. Moreover, in RAEB and RAEBt, a significant correlation was observed between c-mpl, CD34, megakaryocyte glycoprotein IIb (GPIIb) expression, and the presence of dysmegakaryopoiesis. These results indicate that patients with RAEB and RAEBt, with high expression of the c-mpl, CD34, and GPIIb genes, may identify a subgroup of patients with particularly poor prognosis, due to an increased risk of secondary leukemia. More aggressive therapy could be justified in these patients.

Antigens, CD↗

PubMed1995

CDR3 size analysis of T cell receptor V beta transcripts: follow-up study in a patient with T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is generally considered to be a clonal disorder arising as an expansion of committed lymphoid precursors. The generation of functional T cell receptor (TCR) genes involves DNA rearrangement processes. This predisposes immature lymphoid cells to abnormal rearrangements. Previous analysis of the TCR genes strongly suggested the clonal origin of human T-ALL. We present a sensitive clonal analysis of bone marrow TCR V beta transcripts in a case of T-ALL. This study allows the analysis not only of TCR V beta transcripts in tumor cells but also the temporal modification of the global TCR repertoire in the follow-up of clinical specimens from bone marrow aspirates. Moreover, we used clone-specific junctional region oligonucleotide probes corresponding to the clonal leukemic population for the molecular monitoring of the malignant clone throughout the clinical course of the disease. This molecular fingerprint appears to be a sensitive method to detect minimal residual disease. It shows that junctional regions of rearranged TCR beta genes corresponding to the tumor cells can also be detected at the time of the complete remission.

Adult↗

PubMed1995

Detection of malignant B cells in peripheral blood stem cell collections after chemotherapy in patients with multiple myeloma.

Autologous transplantation after high-dose chemo or radiotherapy is now frequently used for the treatment of patients with multiple myeloma (MM). The collection of peripheral blood stem cells (PBSC) has a theoretical advantage over autologous bone marrow collection as the malignant plasmacytic contamination is believed to be lower. However, the extent of B cell contamination in PBSC has not been extensively investigated. Using an immunoglobulin heavy chain gene 'fingerprinting' technique at diagnosis and during apheresis after one cycle of chemotherapy we detected a monoclonal population in 44% of PBSC samples (9 positives in 22 studied). There was no correlation between contamination and sex, age, Durie and Salmon classification, C-reactive protein and albumin. A significant correlation was observed with beta 2 microglobulin serum level (P = 0.02). Twenty one patients were grafted and up to the present, with a mean follow-up of 12 months, 6 patients have relapsed including 4 patients with contaminating B cells. Our results suggest that PBSC contamination, defines a 'poor risk' group of patients, with poor prognosis. However, we could not exclude reinitiation of the disease by plasmacyte stem cells after grafting.

Adult↗

PubMed1995

Expression of the Evi-1 gene in myelodysplastic syndromes.

Increased expression of the proto-oncogene Evi-1 has been shown to block the in vitro granulocytic differentiation of myeloid cells in response to granulocytic colony-stimulating factor and to interfere with the proliferation of erythroid cells in response to erythropoietin. We determined the frequency of Evi-1 expression in myelodysplastic syndromes (MDS), a disorder with altered proliferation and differentiation in the erythroid and myeloid lineages. Twenty-one patients were studied. Abnormal expression was found in 1/9 patients with refractory anemia and in 7/12 patients with refractory anemia with excess of blasts (RAEB) or in transformation (RAEBt). No correlation could be found between expression of Evi-1 and age, sex, hemoglobin level and percentage of bone marrow blasts or erythroblasts. This result suggests that the high incidence of Evi-1 expression which remains at low levels in RAEB and RAEBt is not a major determinant of ineffective erythropoiesis and myelopoiesis in MDS.

Base Sequence↗

PubMed1994

Pattern of expression of five alternative transcripts of the lck gene in different hematopoietic malignancies: correlation of the level of lck messenger RNA I B with the immature phenotype of the malignancy.

The lck gene encodes a tyrosine protein kinase of the src family which is highly expressed in T-lymphocytes. Two widely separated promoters govern expression of the lck gene. We report in this study that alternative splicing between cryptic donor and acceptor sites in the 5' untranslated region of the transcripts initiated at the type I promoter give rise to three different type I lck mRNAs, I A, I B, and I C. Altogether with the types II A and II B reported previously, the lck transcription is thus characterized by five alternative transcripts. We further used the complementary DNA-polymerase chain reaction assay to describe the pattern of expression of these five lck transcripts in different hematopoietic cell lines and in blood or bone marrow samples from healthy donors and leukemic patients. We report that the transcript II A is by far the major transcript present both in human samples and in T-cell lines. The low lck expression in B-cell lines is characterized by the quite exclusive presence of the transcript I B. We show that hematopoietic diseases characterized by the presence of immature cells [acute myeloid leukemia (AML-0 and AML-1) and T- and B-cell acute lymphoid leukemia] exhibit a marked increase of the transcript I B. No significant difference from the normal pattern is observed in AML according to the differentiation stage (AML-2 to AML-5). A normal pattern of lck expression is restored in AML-0 and AML-1 patients at complete remission.

Adolescent↗

PubMed1993

Expression of the c-mpl proto-oncogene in human hematologic malignancies.

Similar to two other hematopoietic growth factor receptors, the c-fms (macrophage colony-stimulating factor receptor) and the c-kit genes, c-mpl has been discovered through the study of oncogenic retroviruses. Unlike c-fms and c-kit, which both belong to a subgroup of tyrosine kinase receptors, the c-mpl proto-oncogene encodes a new member of the cytokine receptor superfamily. We have studied the expression of c-mpl in a series of 105 patients with hematologic malignancies using Northern blot analysis. The levels of c-mpl transcripts in lymphoid malignancies and in chronic myeloproliferative disorders were not significantly different from those found in normal bone marrow cells, in which c-mpl was barely detectable. In contrast, c-mpl expression was increased in 26 of 51 patients with acute myeloblastic leukemia (AML) and in 5 of 16 patients with myelodysplastic syndromes. Amplification of the c-mpl gene was detected in genomic DNA of one M4 AML patient. There was no significant correlation between c-mpl expression and the French-American-British classification of AML. Patients with high c-mpl expression appeared to belong to a subgroup of AML with a low rate of complete remission and a poor prognosis, including secondary leukemia and AML with unfavorable cytogenetic abnormalities.

Acute Disease↗